1996 Abstracts
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Bhat MA, Philp AV, Glover DM and Bellen HJ
Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with Topoisomerase II.
We have isolated a Drosophila gene, barren (barr), required for sister-chromatid segregation in mitosis. barr encodes a novel protein that is present in proliferating cells and has homologs in yeast and human. Mitotic defects in barr embryos become apparent during cycle 16, resulting in a loss of PNS and CNS neurons. Centromeres move apart at the metaphase-anaphase transition and Cyclin B is degraded, but sister chromatids remain connected, resulting in chromatin bridging. This phenotype is similar to that described in TOP2 mutants in yeast. Barren protein localizes to chromatin throughout mitosis. Colocalization and biochemical experiments indicate that Barren associates with Topoisomerase II throughout mitosis and alters the activity of Topoisomerase II. We propose that this association is required for proper chromosomal segregation by facilitating the decatenation of chromatids at anaphase.
Cell 87:1103-1114Glover DM, Ohkura H and Tavares A
Polo kinase: the choreographer of the mitotic stage?
No abstract available.
Journal of Cell Biology 135:1681-1684-
A maternal requirement for glutamine synthetase I for the mitotic cycles of syncytial Drosophila embryos.
We describe the maternal effect phenotype of a hypomorphic mutation in the Drosophila gene for glutamine synthetase I (GSI). The extent of development of embryos derived from homozygous mutant females is variable, although most mutant embryos fail to survive past germband elongation and none develop into larvae. These embryos are characterised by an increase in the number of yolk-like nuclei following nuclear migration to the cortex. These nuclei appear to fall into the interior of the embryo from the cortex at blastoderm. As they do so, the majority continue to show association with PCNA in synchrony with nuclei at the cortex, suggesting some continuity of the synchrony of DNA replication. However, the occurrence of nuclei that have lost cell cycle synchrony with their neighbours is not uncommon. Immunostaining of mutant embryos revealed a range of mitotic defects, ultimately resulting in nuclear fusion events, division failure or other mitotic abnormalities. A high proportion of these mitotic figures show chromatin bridging at anaphase and telophase consistent with progression through mitosis in the presence of incompletely replicated DNA. GSI is responsible for the ATP-dependent amination of glutamate to produce glutamine, which is required in the formation of amino acids, purines and pyrimidines. We discuss how the loss of glutamine could depress both protein and DNA synthesis and lead to a variety of mitotic defects in this embryonic system that lacks certain checkpoint controls.
Journal of Cell Science 109:2649-2660 White-Cooper H, Carmena M, Gonzalez C and Glover DM
Mutations in new cell cycle genes that fail to complement a multiply mutant third chromosome of Drosophila.
We have simultaneously screened for new alleles and second site mutations that fail to complement five cell cycle mutations of Drosphila carried on a single third chromosome (gnu, polo, mgr, asp, stg). Females that are either transheterozygous for scott of the antartic (scant) and polo, or homozygous for scant produce embryos that show mitotic defects. A maternal effect upon embryonic mitoses is also seen in embryos derived from females transheterozygous with helter skelter (hsk) and either mgr or asp. cleopatra (cleo), fails to complement asp but is not uncovered by a deficiency for asp. The mitotic phenotype of larvae heterozygous for cleo and the multiple mutant chromosome is similar to weak alleles of asp, but there are no defects in male meiosis. Mutations that failed to complement stg fell into two complementation groups corresponding to stg and a new gene noose. Three of the new stg alleles are early zygotic lethals, whereas the fourth is a pharate adult lethal allele that affects both mitosis and meiosis. Mutations in noose fully complement a small deficiency that removes stg, but when placed in trans to certain stg alleles, result in late lethality and mitotic abnormalities in larval brains.
Genetics 144:1097-1111Tavares AA, Glover DM and Sunkel CE
The conserved mitotic kinase polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts.
The Drosophila gene polo encodes a protein kinase required for progression through mitosis. Wild-type polo protein migrates as a tight doublet of 67 kDa which is converted to a single band by phosphatase treatment, which also inactivates the kinase. We have determined putative polo substrates in a cell-free system derived from mutant embryos. Exogenous polo protein kinase phosphorylates proteins of sizes 220 kDa, 85 kDa and 54 kDa, to a greater extent when added to extracts of polo1-derived embryos compared with extracts of wild-type embryos, which in both cases have been subject to mild heat treatment to inactivate endogenous kinases. Proteins of the same size are predominantly phosphorylated by the endogenous kinases present in wild-type extracts, and are either not phosphorylated or are poorly phosphorylated in extracts of polo1-derived embryos. We show that a specific monoclonal antibody to β-tubulin precipitates the phosphorylated 54 kDa protein together with an associated 85 kDa protein also phosphorylated by polo protein kinase. Moreover polo binds to an 85 kDa protein which is enriched in microtubule preparations. We discuss the extent to which these in vitro phosphorylation results reflect the effects of mutations in polo on microtubule behaviour during the mitotic cycle.
EMBO Journal 15:4873-4883-
Molecular Immunology (Second edition)
Like other volumes in the Frontiers in Molecular Biology series, this book presents an up-to-date picture of current research topics in a key area. The contributors to this book have been carefully chosen to reflect the main thrusts of research effort across a broad range within the rapidly developing field of immunology. Subjects covered include immunoglobulin gene rearrangement, the MHC antigens, B-cell activation, and the complement system.
Frontiers in Molecular Biology series OUP, Oxford -
DNA Cloning: A Practical Approach, Volume 4 - Mammalian Systems (Second edition)
This is the fourth and last volume in this set. This volume describes a variety of tecnhiques for genetic manipulation of mammalian cells. Topics covered include construction of Vaccinia virus recombinants, retroviral vectors, production of transgenic animals, expression using the HSV-1 vector system, and adenovirus vectors. This book is intended for PhD students, research assistants, postdocs, academic staff, in the general areas of molecular biology, genetics, biochemistry, and medical research.
Practical Approach series OUP, Oxford
